Escherichia coli O157:H7 (E. coli O157:H7) is one of hundreds of strains of the bacterium Escherichia coli. The combination of letters and numbers in the name of the bacterium refers to the specific markers found on its surface and distinguishes it from other types of E. coli. An estimated 73,000 cases of infection and 61 deaths occur in the United States each year. It accounts for about 2% of all cases of diarrhea in the western world, and at least one-third of all cases of hemorrhagic colitis. This bacterium is commonly associated with foods such as ground beef, unpasteurized milk and juice, sprouts, lettuce, salami, and game meat, and contact with cattle. Waterborne transmission occurs through swimming in contaminated lakes, pools, or drinking inadequately chlorinated water. The organism is easily transmitted from person to person and has been difficult to control in child day-care centers. E. coli O157:H7 produces large quantities of one or more related Shiga toxins that cause severe illness and damage in humans. The illness is characterized by severe cramping (abdominal pain) and diarrhea which is initially watery but becomes grossly bloody. Occasionally vomiting occurs. In severe cases, a complication called hemolytic uremic syndrome (HUS), in which the red blood cells are destroyed and the kidneys fail, may develop. (Doyle, P M et al. Food Microbiology, Fundamentals and Frontiers, ASM press, 1997, chapter 10, pp. 171 to 191).
E. coli O157 which are non-motile are designated E. coli O157:H-or O157:NM. E. coli O157:H- or O157:NM are missing the H antigen which is the flagellar or motility antigen. They usually produce verotoxin and cause a similar pattern of disease as E. coli O157:H7.
In order to prevent the occurrence of infections by E. coli O157:H7 and other toxin producing variants such as verotoxin producing E. coli O157:NM, methods of detection can be employed that identify the presence of the bacteria in food, prior to consumer availability and consumption. Many detection techniques, however, require long time periods and, therefore are not time and cost effective due to relatively quick rates of food spoilage. For example, a number of detection technologies require the culturing of bacterial samples for time periods of up to eight days. During that time, however, the product being tested must be placed in circulation for purchase and consumption. Therefore, a system that can rapidly identify the presence of E. coli O157:H7 and other toxin producing variants such as verotoxin producing E. coli O157:NM in food and other test samples is desirable.
A variety of methods are described in the prior art for the detection of bacterial contaminants. One of these methods is the amplification of specific nucleotide sequences using specific primers in a PCR assay. Upon completion of the amplification of a target sequence, the presence of an amplicon is detected using agarose gel electrophoresis. This method of detection, while being more rapid than traditional methods requiring culturing bacterial samples, is still relatively time consuming and subject to post-PCR contamination during the running of the agarose gel.
An additional technology utilized for detection of bacterial contamination, is nucleic acid hybridization. In such detection methodologies, the target sequence of interest is typically amplified and then hybridized to an oligonucleotide probe which possesses a complementary nucleic acid sequence to that of the target molecule. The probe can be modified so that detection of the hybridization product may occur, for example, the probe can be labelled with a radioisotope or fluorescent moiety.
The general use of E. coli nucleic acid sequences for the detection of this bacterium has been described. Many of the described detection methods are specific for certain strains of E. coli, such as O157. Others detect multiple strains of E. coli. For example, U.S. Pat. No. 5,654,417 describes DNA fragments useful for detecting E. coli strains and U.S. Pat. No. 6,365,723 describes genomic sequences, which can be used as diagnostic probes. These sequences are present in E. coli but absent from E. coli K 12. More general methods are provided in U.S. Pat. Nos. 5,693,469 and 6,551,776, which describe hybridization assay probes complementary to E. coli rRNA sequences. In addition to hybridizing to E. coli sequences, these probes hybridize to other genus members and Shigella species.
In addition, a number of PCR based methods of detecting E. coli have been described. For example, U.S. Pat. No. 6,268,143 describes a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. International application WO03/062464A3 describes a kit that has the potential for use directly on foods and environmental samples. The kit comprises three multiplex PCR assays that can detect in E. coli the presence of eight virulence genes: eaeA, EHEC-HlyA, Stx1 (VT1), Stx2 (VT2), Stx2c (VT2c), Stx2d (VT2d), Stx2e (VT2e) and Stx2f (VT2f). While, Desmarchelier et al. (J. Clin. Microbiol. (1998) 36:1801-1804) describe a PCR-based method for detecting E. coli 0157 that involves amplification of a region of the O-antigen synthesis genes followed by gel electrophoresis and Southern blot analysis to confirm the identify of the amplified fragment. The method was capable of identifying two serotypes of E. coli O157; the O157:H7 and 0157:H-serotypes. Another PCR-based protocol based on the amplification of the rfjB region of the O-antigen synthesis genes is described by Maurer et al. (Appl. Environ. Microbiol. (1999) 65:2954-2960).
A useful modification of the above technology provides for the concurrent amplification and detection of the target sequence (i.e. in “real time”) through the use of specially adapted oligonucleotide probes. Examples of such probes include molecular beacon probes (Tyagi et al., (1996) Nature Biotechnol. 14:303-308), TaqMan® probes (U.S. Pat. Nos. 5,691,146 and 5,876,930) and Scorpion probes (Whitcombe et al., (1999) Nature Biotechnol. 17:804-807). The use of TaqMan® probes to detect Escherichia coli in water samples is described by Frahm and Obst in J. Microbiol. Methods (2003) 52:123-131. U.S. Pat. No. 6,664,080 discloses the detection of pathogenic E. coli strains using a Taqman (TM)-PCR based approach comprising the use of primers and fluorogenic probes specific for the genes encoding characteristic virulence factors or toxins.
Molecular beacons represent a powerful tool for the rapid detection of specific nucleotide sequences and are capable of detecting the presence of a complementary nucleotide sequence even in homogenous solutions. Molecular beacons can be described as hairpin stem-and-loop oligonucleotide sequences, in which the loop portion of the molecule represents a probe sequence, which is complementary to a predetermined sequence in a target nucleotide sequence. One arm of the beacon sequence is attached to a fluorescent moiety, while the other arm of the beacon is attached to a non-fluorescent quencher. The stem portion of the stem-and-loop sequence holds the two arms of the beacon in close proximity. Under these circumstances, the fluorescent moiety is quenched. When the beacon encounters a nucleic acid sequence complementary to its probe sequence, the probe hybridizes to the nucleic acid sequence, forming a stable complex and, as a result, the arms of the probe are separated and the fluorophore emits light. Thus, the emission of light is indicative of the presence of the specific nucleic acid sequence. Individual molecular beacons are highly specific for the nucleic acid sequences they are complementary to.
A molecular beacon probe designed to detect the E. coli O157:H7 serotype has been described (Fortin et al., (2001) Analytical Biochem. 289:281-288). The probe was designed to hybridise to an amplified target sequence from the rJbE O-antigen synthesis gene of E. coli O157:H7 that is either 496 base pair (bp) or 146 bp in length, depending on the primers used. The probe was also able to detect E. coli O157:NM and 0157:H-serotypes, but was not intended to detect other strains of E. coli. 
The gnd gene from several bacteria, including many E. coli strains and serotypes, Shigella flexneri, Citrobacter freundii, Citrobacter koseri, Salmonella enterica and Salmonella thyphimurium has been characterized. The gnd gene codes for a decarboxylating gluconate-6-phosphate dehydrogenase (6-PGD) and is a part of the pentose phosphate pathway where it oxidises 6-Phosphogluconate into Ribulose-5-phosphate which is used for the synthesis of nucleic acids. In E. coli the gnd gene is locate in close proximity (200-2000 bp) to the rjb cluster that codes for the O antigen that is used for serotyping Escherichia coli. This advances the concept that new alleles of gnd, created either by point mutation or intragenic recombination, “hitchhike” to high frequency by diversifying selection favoring antigen variation at the adjacent rib locus. In addition, local recombination events involving the rjb region and extending though the gnd locus could result in specific combinations of gnd alleles and rjb genes being cotransferred in nature. The gnd allelle A that was targeted has been found only in E. coli 0157 from the DEC5 lineage [Tarr P I, Schoening L M et al., (2000) Journal of Bacteriology 182(21):6183-9191; Nelson K, Selander R K (1994) Proc. Natl. Acad. Sci. USA. 91:10227-10231].
International Patent Application WO/034484 and U.S. Patent Application 20020150902 disclose the gnd gene sequence of fourteen strains of E. coli (11 of which were E. coli O157:H7) and polymorphisms therein. These applications further disclose that these polymorphisms can be used to identify the presence of a particular strain of E. coli and/or differentiate one strain of E. coli from another, but do not provide a rational approach for the actual detection of specific strains and at the desired level of specificity.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.